Analysis of gene regulation in the vertebrate beta-globin loci has been instructive in establishing many of the founding principles used in differential tissue- and developmental stage-specific transcriptional control. I propose to continue analysis of the trans-acting factors that are employed in differentially regulating the human beta-globin genes, in particular to address the basic mechanisms underlying erythroid tissue- and stage- specific gene regulation (hemoglobin switching). During the previous award period, we discovered and then purified a novel repressor complex (DRED) that suppresses murine embryonic globin gene transcription in adult erythroid cells. The DNA binding scaffold for DRED is composed of a heterodimer formed between two orphan nuclear receptors, TR2 and TR4. Given the clear and immediate potential for therapeutic development for treating hemoglobinopathies, and in particular sickle cell anemia, this discovery should be fully explored. Experiments are proposed here to determine whether the murine and human orthologs of DRED regulate the embryonic and fetal beta-type globin genes in genetic and biochemical experiments to be conducted in loss- and gain-of-function mouse models as well as in primary (CD34+) fetal and adult human erythroid progenitor cells and human embryonic stem cells (HSF-6, WA01 and BG01). We will also determine the identity of other co-repressor constituents of the DRED complex using in vivo biotin affinity labeling followed by tandem mass spectrometry; these co- effectors can also be developed as therapeutic targets for inactivation that may lead to adult erythroid gamma- (or epsilon-) globin gene induction.